PROJECT SUMMARY Endogenous DNA damage can contribute to aging, sporadic cancers, and neurodegeneration. There are many sources of endogenous DNA damage, including oxidation, alkylation, nucleobase deamination, and hydrolysis of the glycosidic bond to generate abasic sites. DNA repair systems mitigate the effects of these endogenous damages, but some lesions inevitably evade repair with deleterious consequences. Importantly, not all DNA damage is created equal. Interstrand cross-links are exceptionally bioactive lesions because they block the DNA strand separation required for read-out and replication of genetic information in cells. Cells have no good answer to cross-links in their genome: Failure to repair cross-links may lead to cell death, tissue dysfunction, and aging, while error-prone repair may result in mutagenesis and cancer. The evolution and retention of elaborate cross-link repair systems across all walks of life suggest that the generation of endogenous interstrand cross-links is an unavoidable fact of life, but the identities of these cross-links and their biological consequences remain uncertain. The long-term objective of this application is to assess the occurrence and biological endpoints of endogenous DNA cross-links, which will contribute significantly to overall understanding of cancer etiology. Work during the previous grant period characterized a group of interstrand cross-links derived from an abasic (Ap) site that is the most common endogenous lesion found in cellular DNA. The work further developed LC-MS methods for the detection and characterization of these lesions in duplex DNA, and described a shuttle vector method for assessing the efficiency and fidelity with which the Ap-derived cross-links are repaired and replicated in human cells. The proposed work, will (1) conduct unbiased screens to comprehensively determine the sequence hotspots for Ap-derived cross-link formation, (2) detect the occurrence of Ap-derived cross-links in cellular DNA, and (3) determine the efficiency and fidelity with which Ap-derived cross-links are repaired in human cells and define the mechanisms of replication-dependent cross-link ?unhooking? in vitro and in human cells. The proposed work explores the hypothesis that formation and replication-coupled repair of Ap-derived cross-links may be particularly important in cancer, neurodegeneration, and aging. The work is significant because the formation and repair of endogenous DNA cross-links may contribute to the causal processes involved in aging and early carcinogenesis. The work is innovative because it examines structurally novel cross-links and novel repair mechanisms, thus promising novel insights regarding the roles of endogenous DNA damage in human health and disease. In the long run, the results may enable understanding of how genetic differences in cross-link repair capacity contribute to human healthspan. Ultimately such insights could inspire approaches that improve health by genetic means or by pharmacological agents that inhibit the formation and/or enhance the repair of endogenous cross-links.